CN108291243A

CN108291243A - The method for producing l-methionine

Info

Publication number: CN108291243A
Application number: CN201680068472.8A
Authority: CN
Inventors: I·奥克尤贝尔; D·菲舍尔; B·巴特; M·哈塞尔迈尔; M·汉佩尔; J·佩达尔
Original assignee: Evonik Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2015-11-27
Filing date: 2016-11-01
Publication date: 2018-07-17
Also published as: RU2018123145A3; EP3380627A1; RU2018123145A; MY186059A; AR106834A1; US20180346946A1; PL3380627T3; BR112018010747A8; EP3380627B1; KR20180086240A; ES2748226T3; MX2018006434A; RU2729012C2; WO2017089077A1; BR112018010747A2; US11034985B2

Abstract

The present invention relates to a kind of methods producing L methionines, wherein microorganism is cultivated under conditions of there are the salt or dimethyl disulphide of L homoserine and methyl mercaptan, methyl mercaptan, to accumulate L methionines in the medium.

Description

The method for producing l-methionine

The present invention relates to a kind of methods producing l-methionine, wherein there are L- homoserine and methyl mercaptan, first sulphur Microorganism is cultivated under conditions of the salt or dimethyl disulphide of alcohol, to accumulate l-methionine in the medium.

Currently, amino acids methionine is worldwide by industrialized production in large quantities, and there is comparable business Importance.Methionine can be used for many fields, such as pharmacy, health and fitness product, but especially as feed addictive For in many feeds of various livestocks, wherein the methionine of racemic modification and pure enantiomeric form to can be used.

At industrial scale, methionine is generated by Bucherer-Bergs reactive chemistries, it is Strecker The variant of synthesis.In this case, initial substance methyl mercapto propionic aldehyde (being prepared by methacrylaldehyde and methyl mercaptan), hydrogen cyanide, ammonia Obtain 5- (2- methyl mercaptos ethyl) hydantoins (methionine hydantoins) with carbon dioxide reaction, then by basic hydrolysis with Alkali metal methionine salt is provided, and then discharges methionine (0 780 370A2 of EP) by being neutralized with acid.May be used also Methionine is prepared using various other methods, such as the hydrolysis of amide carbonylation reaction, protein or microbial fermentation generate Methionine.In chemical synthesis, methionine is generated as D- and the racemic mixture of l-methionine, wherein such as L- The precursor or L- homoserine of methionine or its L- configuration can be generated by suitable microbial fermentation.

L- homoserine is the potential precursor of l-methionine (HJ Teas et al., J.Biol.Chem.1948,172： 651-658), can be generated by chemical mode (MD Armstrong, J.Am.Chem.Soc., Vol.70,1756-1759, 1948) and the mode of microorganism ferments generation (see, for example, US 3,598,701, US 6,303,348B1, EP 0994190A2, EP 1149911A2, WO 2004/067757A1).

Hateley et al. discloses the method for obtaining l-methionine by the chemistry route since L- homoserine (WO 2007/085514 A2)。

Lievense is able to demonstrate that the microbial strains for lacking homocysteine methyl enzymatic activity, (big with original strain Enterobacteria, Corynebacterium glutamicum or yellow bacillus) it compares, L- homoserine acetyltransferases and O- second are encoded by conversion The plasmid of acyl-L- sulfhydrylases (O-acetylhomoserine (mercaptan)-lyase), generates in the presence of methyl mercaptan It is more than the l-methionine of its own needs, L- homoserine yield raises (93/17112 A1 of WO).

Bolten et al. (J.Microbiol.Biotechnol. (2010), 20 (8), 1196-1203) is able to demonstrate that (wild Raw type) Corynebacterium glutamicum (C.glutamicum) can be in methyl mercaptan and its dimer (dimethyl disulphide) as unique Sulphur source in the case of growth without sulfate (it is the sulphur source for cultivating microorganism most normal See), and have studied potential logical Road and enzyme.They prove, the responsible methyl mercaptans of MetY (O- acetyl-L- sulfhydrylases) or dimethyl disulphide Sulfydryl substitution O- acetyl-L- homoserine Acetyl Groups directly to generate l-methionine.In order to increase L- first sulphur ammonia The yield of acid, the authors suggested that not only to expand MetY, but also will expand other enzymes of l-methionine biosynthesis.

Zelder et al. (2007/011939 A2 of WO) proves that l-methionine can be in microorganism by being sealed in methyl Culture has the O- second for releasing regulation and control in the presence of the sulphur compound (such as dimethyl disulphide or dimethyl trisulfide) at end Acyl-L- sulfhydrylases or the transfer of O- succinyl-L sulfhydrylases and/or L- homoserine acetyl The microorganism (such as Escherichia coli (E.coli) and Corynebacterium glutamicum) of enzyme or L- homoserine succinyltransferases produces It is raw.

Kim et al. (2008/013432 A1 of WO) proposes the two benches biotechnology side for being used to prepare l-methionine Method.In first step reaction, L-methionine precursor, O- succinyl-L- homoserine or O- acetyl-L- homoserine are initial It is obtained by the method for recombinant microorganism, they are accumulated in culture solution.In next second step, with O- ambers In the presence of acyl-L- sulfhydrylases activity or the active protein of O- acetyl-L- sulfhydrylases, Express these protein microorganism or the microorganism cell dissociation object in the presence of, L-methionine precursor and first sulphur Alcohol is reacted to provide l-methionine and corresponding carboxylic acid (i.e. acetic acid or succinic acid).

However, in the enzymatic reaction, other than l-methionine, the acetic acid or amber of equimolar amounts are yet formed Acid.For example, when selecting O- acetyl-L- homoserine as L-methionine precursor, during the reaction (especially in industry In the scale of change) lead to high acetic acid concentration.Under external low ph value, the molecular acid that does not dissociate may pass through film enter cell and Wherein by deprotonation, this cause the pH inside cytoplasm to decline and upset cellular pH stable state (IRBooth, Microbiological Reviews 49, No (1985), 359-378).Additionally, it is difficult to by acetic acid with acceptable cost (effort) it is removed completely from l-methionine product.Therefore, Hong et al. (2012/091479 A2 of WO) proposes many Method generates in second stage during l-methionine generates from removing and recycle in l-methionine product Relatively great amount of acetic acid.

The method that the object of the present invention is to provide a kind of to generate l-methionine in microorganism, wherein in O- acetyl-L- Homoserine is converted to the acetic acid formed in l-methionine and is substantially recycled by identical microorganism.

The purpose can be realized by the following method for generating l-methionine, wherein including L- homoserine and sulphur source Culture medium in culture with L- homoserine O- acetyltransferase activities and O- acetyl-L- sulfhydrylases activity Microorganism, the sulphur source is selected from：Methyl mercaptan (MC), the salt of methyl mercaptan and dimethyl disulphide (DMDS), are thus being cultivated L-methionine is accumulated in base.

Enzymatic activity in microorganism is usually realized by the expression of the corresponding gene of the corresponding enzyme of coding.So-called promoter Positioned at the upstream of gene.The DNA sequence dna that promoter is made of about 40 to 50 base-pairs, and it constitutes rna polymerase holoenzyme Binding site and transcripting start point (M.P á tek et al., Microbial Biotechnology, 6 (2013), 103-117), Thus the expression intensity of controlled polynucleotides or gene can be influenced." functionality connection ", it is intended that promoter and gene it is continuous Arrangement, leads to the transcription of the gene.

Microorganism can also be recombination, and the O- of L- homoserine O- acetyltransferase activities and enhancing with enhancing Acetyl-L- sulfhydrylase activity.

The enzymatic activity enhanced in microorganism is may be implemented in for example, by the mutation of corresponding endogenous gene.Enzymatic activity can also pass through Increase the expression of corresponding gene, for example, enhancing enzyme activity by increasing gene copy number and/or by enhancing the gene regulation factor Property.The enhancing for having positive these regulatory factors influenced on gene expression, can be for example, by the startup of modification structure upstream region of gene Subsequence is realized to enhance the effect of promoter or by replacing the promoter completely with more effective promoter.

In the method according to the invention, L- homoserine O- acetyltransferase activities and O- acetyl-L- homoserine sulphur Change hydrogenolysis enzymatic activity preferably has the protein of L- homoserine O- acetyltransferase activities by enhancing coding, or has The expression of the gene of the active protein of O- acetyl-L- sulfhydrylases and enhance.The gene expression of enhancing is preferred Vulcanize by protein of the increase coding with L- homoserine O- acetyltransferase activities or with O- acetyl-L- homoserine The copy number of the gene of the active protein of hydrogenlyase is realized, and/or passes through functional connection coding tool in each case There is the protein of L- homoserine O- acetyltransferase activities or there is the active egg of O- acetyl-L- sulfhydrylases It is realized on the gene to strong promoter of white matter.

Suitable strong promoter generates this promoter to increase the method for expression, be in the literature it is known (such as S.Lisser and H.Margalit,Nucleic Acid Research,1993,Vol.21,No.7,1507-1516；M.Pá tekand J.Nesvera in H.Yukawa and M Inui(eds.),Corynebacterium glutamicum, Microbiology Monographs 23,Springer Verlag Berlin Heidelberg 2013,51-88； B.J.Eikmanns et al.,Gene,102(1991)93-98).It is connect with these promoter functions for example, being increased by For gene expression, natural promoter can be optimized by changing promoter sequence on the direction of known consensus sequence (M.Patek et al.,Microbiology(1996),142,1297-1309；M.Patek et al.,Microbial Biotechnology 6(2013),103-117).In order to increase egg of the coding with L- homoserine O- acetyltransferase activities The gene of gene (metX) or coding with the active protein of O- acetyl-L- sulfhydrylases of white matter (metY) expression, for example, tacI promoters (PtacI) be it is applicable (H.A.deBoer et al., Proc.Natl.Acad.Sci.USA,Vol.80,21-25,January 1983,Biochemistry).(the SEQ ID of sequence number 5 No.5 the sequence of PtacI) is shown.

Constitutive promoter is also applied for being overexpressed, and wherein the active gene of codase can continuous table under promoter control It reaches, such as glucose dependency deo promoters.The promoter of chemical induction type is also applicable, such as tac, lac or trp.With In the most common system of inducible promoter be the lac operons of Escherichia coli.In this case, using lactose or isopropyl Base β-D- thio-galactose pyran-glucosides (IPTG) are used as derivant.Similarly, using arabinose (such as pBAD systems) or Rhamnose (such as Escherichia coli KRX) is common as the system of derivant.For the system of physics inducement type, such as based on The cold shock promoters system of the temperature inducible of Escherichia coli cspA promoters from Takara or Lambda PL, and Permeate inducible promoter, such as osmB (such as WO 95/25785A1).

In the method according to the invention, recombinant microorganism is selected from：Enterobacteriaceae (Enterobacteriaceae) and stick Bacteriaceae (Corynebacteriaceae), such as Escherichia coli (E.coli) bacterial strain, such as avirulence e. coli k-12 Bacterial strain MG1655 (DSM 18039) or Corynebacterium glutamicum (Corynebacterium.glutamicum) bacterial strain, such as ATCC13032 or Corynebacterium humireducens (C.humireducens) bacterial strain, such as DSM 45392.

In the method according to the invention, L- homoserine O- acetyltransferase activities are such as MetX enzymes, are originated from paddy Propylhomoserin bar bacterium comes from C.humireducens.Kim et al. (2 657 345 A1 of EP；2 657 250 A2 of EP) or Ochrombel et al. (2015/165746 A1 of WO) is disclosed with the suitable of L- homoserine O- acetyltransferase activities The example of enzyme.The MetX enzymes used in following EXPERIMENTAL EXAMPLESThes have the amino acid sequence of sequence number 2 (SEQ ID No.2). Sequence number 1 (SEQ ID No.1) shows the corresponding nucleotide sequence of gene metX.The sequence derives from Corynebacterium glutamicum (ATTC13032)NC_003450。

Suitable for O- acetyl-L- sulfhydrylase activity according to the method for the present invention, it is e.g. originated from paddy ammonia The MetY enzymes of sour bar bacterium or C.humireducens.Et al. (WO 02/18613A1),Et al. (WO 2007/024933 A2) or Kim et al. (2 657 345 A1 of EP) disclose and according to the present invention there is O- acetyl-L- Kosé The example of the active enzyme of propylhomoserin sulfhydrylase.The MetY enzymes used in following EXPERIMENTAL EXAMPLESThes have according to sequence number 4 The amino acid sequence of (SEQ ID No.4).Sequence number 3 (SEQ ID No.3) shows the corresponding nucleotide sequence of metY genes. The sequence derives from Corynebacterium glutamicum (ATTC13032) NC_003450.

L- homoserine is transported to by the input albumen (importer) of branched-chain amino acid in microorganism, for example, large intestine In bacillus LIV systems (B.A.Temptonton and M.A.Savageau, JOURNAL OF BACTERIOLOGY, Vol.117, No.3, in March, 1974, the 1002-1009 pages).In Corynebacterium glutamicum, also by the homologous of the BrnQ of cgl2310 codings Movement system (A.Tauch etc., Arch Microbiol 169 (1998)：303-312).

In the cell, by the acetyl grouptransfer of acetyl coenzyme A (acetyl-CoA) to pass through (heterologous) homoserine O- second Acyltransferase (MetX) provides the hydroxy activated of L- homoserine to O- acetyl-L- homoserine.Then O- acetyl-L- Kosé ammonia Acid passes through (heterologous) sulfhydrylase in the presence of restoring sulphur source (such as methyl mercaptan (MC) and 5 '-phosphopyridoxal pyridoxal phosphates (PLP)) (MetY) l-methionine and acetic acid are converted to.Although O- acetyl-L- homoserine is that methionine biosyn synthesizes in bar bacterium One of natural intermediate, but biosynthesis of the methionine in enterobacteriaceae with by O- succinyl-L- homoserine Mesosome carries out similar (for example, see Figge R (2007) Methionine biosynthesis in Escherichia coli and Corynebacterium glutamicum.In:Wendisch VF(ed)Amino acid biosynthesis- Pathways, regulation and metabolic engineering.Microbiology Monographs, volume 5, 163-193 pages of Springer, Berlin, the).Therefore it may first have to by L- homoserine O- transacetylases and O- acetyl-L- It is introduced into sulfhydrylase active heterologous in enterobacteriaceae (such as Escherichia coli), and these enzymatic activitys are naturally occurring In bar bacterium (such as Corynebacterium glutamicum).Encoding the corresponding homologous or heterologous gene of corresponding enzyme respectively can be described by starting Method (such as increase the copy number of two kinds of genes and/or use strong promoter) enhances.

In enterobacteriaceae such as Escherichia coli, L- homoserine O- transacetylases and the vulcanization of O- acetyl-L- homoserine The active enhancing of hydrogenlyase can be introduced by way of converting suitable carrier, and the suitable carrier includes gene order metX (such as SEQ ID No.1) and metY (such as SEQ ID No.3), in each case be located at strong promoter (such as PtacI upstream).The example of this construct is the sequence according to sequence number 6 (SEQ ID No.6).

In the presence of methyl mercaptan (MC) and 5 '-phosphopyridoxal pyridoxal phosphates (PLP) and (heterologous) sulfhydrylase (MetY), lead to It crosses the acetic acid for discharging O- acetyl-L- homoserine to the conversion of l-methionine and then is again used to induce by acetic acid Type Acetyl-CoA synthetase (Acs) consumes ATP synthesis acetyl-in the cytoplasm of Escherichia coli (bacillus subtilis is also the same) CoA, the Acetyl-CoA synthetase are especially conditioned object CsrA activation (S.Kumari et in stationary phase or under anaerobic al.,JOURNAL OF BACTERIOLOGY,Vol.177,No.10,May 1995,p.2878–2886)。

Compared to Escherichia coli, the excessive acetic acid in Corynebacterium glutamicum is by acetokinase (AK) in ATP dependences Acetyl phosphate is specifically converted in reaction, finally by phosphate transacetylase (phophotransacetylase, PTA) Reaction provides acetyl-CoA in the presence of CoA.In Corynebacterium glutamicum, corresponding gene, ack and pta are arranged at one By (R.Gerstmeir et al.Journal of Biotechnology in the operon of acetic acid adjusting on transcriptional level 104(103)99-122)。

Albumen (exporter) is exported by YjeH, and (Q.Liu et are discharged in l-methionine from Bacillus coli cells al.,Appl Environ Microbiol 81(2015)p.7753–7766).In addition, the gene ygaZH in Escherichia coli is compiled The output albumen (WO 2015/028675A1) of code methionine.By means of BrnFE output albumen by l-methionine from paddy ammonia (C. is discharged in culture medium in the cell of sour bar bacteriumet al.,JOURNAL OF BACTERIOLOGY,June 2005,p.3786–3794)。

Fig. 1：Show pMW218 plasmid maps.

Fig. 2：Show pMW218_Ptac-metX_Ptac-metY plasmid maps.

Fig. 3：Show the cell homogenates pair of MG1655/pMW218 or MG1655/pMW218_Ptac-metX_Ptac-metY The catalyzed conversion of L- homoserine and acetyl-CoA.

Fig. 4：It is shown in the presence of O-acetylhomoserine and 5 '-phosphopyridoxal pyridoxal phosphates (PLP) through O- acetyl Kosé ammonia Sour sulfhydrylase (MetY) catalyzed conversion methyl mercaptan sodium.Show MG1655/pMW218 and MG1655/pMW218_Ptac- Comparison of the cell homogenates of metX_Ptac-metY under used corresponding total protein concentration.

Embodiment

1) the L- homoserine O- transacetylases of heterogenous expression bar bacterium species and the gene of sulfhydrylase are prepared Enterobacteria

Based on the genome sequence of Corynebacterium glutamicum (ATCC13032) NC_003450, composite coding is respectively provided with SEQ The L- homoserine O- transacetylases of ID No.2 amino acid sequences and the O- acetyl-with SEQ ID No.4 amino acid sequences The gene order metX (SEQ ID No.1) and metY (SEQ ID No.3) of L- sulfhydrylases, both have Upstream promoter PtacI (SEQ ID No.5) from Life Technologies Invitrogen GeneArt (Germany) (H.A.deBoer etc., Proc.Natl.Acad.Sci.USA, volume 80, the 21-25 pages, January nineteen eighty-three, biochemistry) (SEQ ID No.6)。

In the SEQ ID No.6, PtacI promoters are that the gene order of base-pair 407-447, metX are 502- 1638 and be that PtacI promoters are 1645-1685 and the gene order of metY is 1742-3055.

Then, pass through carrier sequence pMW218 (accession number：AB005477) in (Nippon Gene, Toyama, Japan) Restriction site BssHII and BglI carry out the clone (Fig. 1) of the composition sequence.Plasmid pMW218_Ptac-metX_Ptac- MetY is formed (Fig. 2) by it.In order to analyze pMW218_Ptac-metX_Ptac-metY plasmids, pass through Eurofins MWG Operon is additionally carried out DNA sequencing.The DNA sequence dna of acquisition is carried out using clone's management (Clone Manager) software correct Property inspection, with confirm nucleotides sequence row be SEQ ID No.6.

Plasmid pMW218 and pMW218_Ptac-metX_Ptac-metY are transformed into e. coli k-12 bacterial strain respectively In MG1655 (DSM No.18039).Then by transformant on the LB medium agar tablets containing 50 μ g/ml kanamycins Culture, so as to generate MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains.It selects respectively It is inoculated into the LB culture mediums that 10ml contains 50 μ g/ml kanamycins, and is trained at 37 DEG C of 200rpm by one bacterium colony respectively It supports 6 hours.Then, 200 μ l growth cell cultures are inoculated into 10ml culture medium As [25g/l ammonium sulfate；1g/l seven is hydrated sulphur Sour magnesium；2g/l potassium dihydrogen phosphates；Seven hydrated irons of 0.03g/l；0.02g/l Manganous sulfate monohydrates；Mono- glucose monohydrates of 20g/l； 30g/l calcium carbonate；0.05g/l kanamycins；0.025g/l 5 '-phosphopyridoxal pyridoxal phosphates (PLP)；0.0024g/l isopropyl-β-D- Thio-galactose pyran-glucoside (IPTG)], and incubated 16 hours at 37 DEG C of 200rpm.It is fresh with 10ml in 100ml flasks It is 2 that these cell cultures are diluted to OD by culture medium A, and is further cultivated under the same conditions until it is about 5 to reach OD values (about 3-4 hours).Then, it is in exponential phase of growth and with homoserine O- transacetylases (MetX) and vulcanization hydrogenolysis These active cells of enzyme (MetY) can be used for bioconversion.Bioconversion is understood to that substance converts, wherein using complete Living cells, fixed cell or separation resolvase or the enzyme or combinations of the above that are connect with carrier.

2) enzymatic activity of L- homoserine O- transacetylases and acetyl-L- sulfhydrylases is detected

It is connect respectively with the single bacterium colony of MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains Kind 10ml contains the LB culture mediums of 50 μ g/ml kanamycins, and is cultivated 6 hours at 37 DEG C of 200rpm.Then, with 200 μ l growths Bacterial cultures is inoculated into 10ml culture medium As (referring to embodiment 1), and is incubated 16 hours at 37 DEG C of 200rpm.Then distinguish Cell culture (8ml is normalized to OD=1) is harvested, supernatant is removed for (20 minutes, 4000rpm, 4 DEG C) by centrifugation, and will The cell of precipitation is cleaned twice with 800 μ l 0.1M kaliumphosphate buffers (pH7.5), and is dissolved in 1ml buffer solutions. Mechanical clasmatosis is carried out in FastPrep FP120 instruments (QBiogene, Heidelberg), wherein by cell with 300mg beadesDigestion vessel in point with 6.5m/s shake three times, 20 seconds every time.Then by crude extract with 12000rpm is centrifuged 20 minutes at 4 DEG C, to remove indigested cell and cell fragment.Use Bio-Rad quantification of protein Measuring method (Bio-Rad, USA) measures the total amount of protein.Then cell homogenates is used for cytoplasm L- homoserine O- acetyl Transferase and the active enzyme process detection of acetyl-L- sulfhydrylases.

2a) detect MetX (the L- homoserine O- transacetylases) activity in cytoplasm

The reaction of L- homoserine O-acetyls transferase (MetX) [EC2.3.1.31] catalysis is by L- homoserine It is O- acetyl-L- homoserine and CoA with acetyl-CoA.By DTNB solution (5,5 '-two thiobis -2- nitrobenzoyls Acid, " Ellmans reagents ", Sigma Aldrich, Germany), the process of the reaction can be by measuring the absorbance at 412nm It records, because the SH groups of DTNB and coacetylase (CoA) can form yellow substance (S.Yamagata Journal of Bacteriology 169, No.8 (1987) 3458-3463).The absorbance measurement of MetX enzymes carries out at 37 DEG C, in advance Using between 0-200 μM the CoA of concentration calibrated.Each preparation containing 100mM kaliumphosphate buffers (pH7.5), 0.65mM DNTB [100 μ l 1.3mM DTNB stostes], 0.13mM acetyl coenzyme As [30 μ l 0.886mM acetyl coenzyme A stostes, Sigma Aldrich, Germany], 10mM L- homoserine [the 100mM L- homoserine stostes of 20 μ l, Sigma Aldrich, Germany] and each cell homogenates specific protein concentration 0.012mg/ml or 0.024mg/ml 0.2ml reaction systems in carry out.

Since acetyl coenzyme A is used for various biosynthesis in the cell, so being urged there are a variety of enzymes in cytoplasm Change acetyl coenzyme A and cut into coacetylase, thus needs to consider the difference between the cell homogenates with and without MetX.

The observation of enzymatic determination result is shown, the cell homogenates of MG1655/pMW218_Ptac-metX_Ptac-metY DNTB absorb increase MG1655/pMW218 (Fig. 3) is consistently higher than in entire time course.Which demonstrate MetX works here There is catalytic activity for additional enzyme.The slope in the initial linear region of institute's recording curve compares display, MG1655/ The activity of every gram of gross protein is about 580 μm of ol/min in the cell homogenates of pMW218, and MG1655/pMW218_Ptac- The activity of every gram of gross protein is about 730 μm of ol/min in metX_Ptac-metY.Therefore, which is L- homoserine O- second The specific activity (specific activity) of acyltransferase (MetX), the about 150 units/g gross proteins (μ of 1 unit=1 Mol substrates conversion/minute).

2b) detect MetY (O- acetyl-L- sulfhydrylases) activity in cytoplasm

The reaction of O- acetyl-L- sulfhydrylases (MetY) [EC 2.5.1.49] catalysis is in 5 '-phosphoric acid pyrroles Tremble in the presence of aldehyde (PLP) conversion O- acetyl-L- homoserine with methyl mercaptan (MC) to provide l-methionine and acetic acid.Strictly according to the facts Apply described in a 2a, since DTNB is reacted with the SH groups of unreacted methyl mercaptan and generate yellow substance, can by DTNB at 412nm absorbs the method for measurement of average value to measure the progress of the reaction.For this purpose, by two bacterial strain MG1655/ PMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY makes cell homogenates thing as described above, and subsequent The reduction or conversion of substrate methyl mercaptan sodium are measured in enzymatic determination.

Each is prepared carries out in the 1ml reaction mixtures at 37 DEG C, and the 1ml reaction mixtures contain 100mM phosphoric acid Potassium buffer solution (pH7.5), 2mM methyl mercaptans sodium (NaMC) [the 200mM NaMC stostes of 10 μ l], 3mM OAHHCl be [30 μ l's 100mM OAH HCl stostes] and 0.01mM PLP [the 1mM PLP stostes of 10 μ l] and corresponding cell homogenates (its gross protein Concentration is respectively 0.012g/l, 0.024g/l or 0.048g/l).After enzyme reaction in limited time, by DTNB to NaMC contents into Row photometric measurement, wherein the MC using concentration between 0-200 μM is calibrated in advance.For this purpose, by the DTNB solution of 180 μ l (4mg/ml) is added in every part of 20 μ l enzymatic reaction mixtures, and is then measured at 412nm.

Due to the enzymatic activity of MetY, total protein concentration, MG1655/pMW218_Ptac-metX_Ptac-metY are depended on It is even in the cell of MG1655/pMW218 bacterial strains that the presence of the cell homogenates of bacterial strain causes the reduction for the NaMC being catalyzed to be significantly faster than The reduction (Fig. 4) of NaMC in the presence of slurry.It is equally occurring but more in the cell homogenates prepared product of MG1655/pMW218 Weak NaMC reduces unrelated with the amount of protein used.Identical drop is also observed that in the prepared product of not cell homogenates It is low, and this is because the chemical dependencies of the methyl mercaptan generated from solution are discharged.According to the slope difference in the range of linearity, can count The specific activity for calculating MetY sulfhydrylases is about 1500 units/g gross proteins (1 unit=1 μm ol substrates conversion/minute), It is in phase same level with MetX enzymes.

3) the intracellular bioconversion of L- homoserine and methyl mercaptan sodium is detected to provide l-methionine.

Culture MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterium as described in example 1 above Strain, and OD600 is then adjusted to about 7 in the exponential phase of respective prepared product.

Then in 100ml shaking flasks, bioconversion, time 0,2,4 and 24 hours are carried out under 37 DEG C of 200rpm.Each Preparation carries out in 10ml culture medium As, and culture medium A contains 6.5g/l L- homoserine, and [the 100g/l homoserine of 500 μ l is former Liquid] (Sigma Aldrich, Germany), 3g/l NaMC [the 6%NaMC stostes of 500 μ l] and 12g/l KH₂PO₄[600 μ l's 200g/l KH₂PO₄Stoste].

The conversion of L- homoserine and methyl mercaptan is carried out using MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains To generate l-methionine, and l-methionine (table 1) is not synthesized using MG1655/pMW218 bacterial strains.Based on what is be initially added The various yields of the amount of NaMC and the amount of L- homoserine based on consumption are based on the presence of two kinds of substrates when starting but then The abiogenous evaporation of methyl mercaptan etc. stoichiometries (equal stoicjiometry).

Table 1：Using MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains, compare The bioconversion of the L- homoserine and 3g/l sodium methyl mercaptides of 6.5g/l synthesis.It shows based on NaMC pulses when reacting beginning Amount and L- homoserine (L-HS) consumption amount, at any time process obtain l-methionine titre and relative yields.

In addition, bioconversion has been carried out using MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains, wherein using Deuterate NaMC (D₃CSNa the NaMC stostes from Sigma Aldrich) are replaced.This is by by CD₃SD (Sigma-Aldrich, 98 atom %D) sodium hydrate aqueous solution of equimolar amounts is introduced to prepare.(alternatively, can according to J.Voss et al., Phosphorous, Sulfur and Silicon and the Related Elements, 2012,187,382, with thiocarbamide and CD₃Prepared by I).The solution after reaction in 24 hours is analyzed by LC-MS, shows the ratio of methionine and methionine-d-3 It is 1：200.Therefore it can detect, the methionine formed in bioconversion is largely the methyl mercaptan by being incorporated to external supply It is formed by.

4) the L- homoserine generated by fermentation is converted by l-methionine by bioconversion

The L- homoserine synthesized based on embodiment 3a is investigated to the bioconversion of l-methionine and passes through hair The bioconversion for the L- homoserine that ferment generates.A concentration of 10g/l for the L- homoserine culture mediums that fermentation generates.According to implementation Example 1 cultivates MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains, and in the 5g/l L- homoserine of fermentation generation In the presence of carry out bioconversion when exponential phase OD value is 5 and carry out 2,4 and 24 hours as shown in embodiment 3a.As shown in table 2, About 7% after bioconversion 2 hours, about 12% and about 45% after bioconversion 24 hours after bioconversion 4 hours Substrate L- homoserine or NaMC are converted into l-methionine, are about 2.9g/l l-methionine which reflects maximum titre.

Table 2：The 5g/l L- high generated that will ferment are passed through by MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains Serine and 3g/l methyl mercaptan sodium carry out the l-methionine and relevant volume of bioconversion formation.

Time(h)	0	2	4	24
					L-Met(g/l)	<0.005	0.45	0.79	2.87
Ferment the L- homoserine (g/l) generated	5.26	4.74	4.24	2.15
					The NaMC (mol/mol) that L-Met/ is initially put into	0%	7%	12%	45%
The L- homoserine (mol/mol) of L-Met/ consumption	0%	69%	62%	74%

5) cell of acetic acid recycles during bioconversion

The amount of the acetic acid formed to study the bioconversion by L- homoserine and methyl mercaptan, in 5g/l L- Kosé Propylhomoserin and 3g/l methyl mercaptans sodium and 12g/l KH₂PO₄[the 200g/l KH of 600 μ l₂PO₄Stoste] in the presence of, it uses Prepared by MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains, contain for their acetic acid Amount records described prepare 4 hours.

MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterium are prepared as described in example 1 above Strain, therefore as described in embodiment 3b, the exponential phase cultures that OD values are about 3 will be originated respectively and be used in 100ml flasks 10ml prepared products.

It is had recorded in table 3 through MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains The acetic acid concentration formed during bioconversion 5g/l L- homoserine and 3g/l methyl mercaptan sodium.L-methionine only with It is formed in the prepared product of the bacterial strain of heterogenous expression metX and metY gene, and in the prepared product of control strain MG1655/pMW218 In cannot detect l-methionine.

Within first four hour, due to the experiment parameter in control prepared product, about 11mM acetic acid is formed, and in bioconversion In form about 17mM acetic acid and 7mM l-methionine.Therefore the excess of the acetic acid measured in bioconversion prepared product is led Cause the difference of 6mM.Since the generation of the equimolar of acetic acid and methionine has synthesized additional methionine, in Cell-free system It did not describe (WO 2008/013432A1), which is 7mM.Therefore, it can detect in bioconversion, l-methionine is closed At the intracellular recycling of the acetic acid of middle formation.So the additional acetic acid generated by bioconversion, it is clear that pass through acetyl-CoA Synzyme (Acs) is partly recycled to acetyl-CoA.

Table 3：In the presence of 5g/l L- homoserine and 3g/l methyl mercaptan sodium, with MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains form in bioconversion prepared product and recycle acetic acid.

6) external to add L- homoserine

Method as described in example 1 above prepares MG1655/pMW218 and MG1655/pMW218_Ptac-metX_Ptac- MetY bacterial strains, therefore as described in embodiment 3b, the exponential phase cultures that OD values are about 3 will be originated respectively and be used in 100ml flasks In 10ml prepared products.After 0h time points (initially no added), the L- Kosé ammonia of 5g/l is added in respective prepared product Acid, addition 3g/l methyl mercaptans sodium and 12g/lKH₂PO₄[the 200g/l KH of 600 μ l₂PO₄Stoste] and add 5g/l L- Kosé Propylhomoserin and 3g/l methyl mercaptans sodium and 12g/lKH₂PO₄[the 200g/l KH of 600 μ l₂PO₄Stoste].

The potency of l-methionine and L- homoserine in 0,2,4 and 6 hour these time point determining prepared product.

Table 4：When containing and being free of 3g/l methyl mercaptans sodium (Na-MC), to using MG1655/pMW218 and MG1655/ Outside pMW218_Ptac-metX_Ptac-metY bacterial strain bioconversions prepared product after addition 5g/l L- homoserine (L-HS) L-methionine and L- homoserine titre.

7) bioconversion or L- homoserine and methyl mercaptan sodium of L- homoserine and dimethyl disulphide (DMDS) (NaMC) bioconversion is to provide l-methionine

Culture MG1655/pMW218_Ptac-metX_Ptac-metY bacterial strains as described in example 1 above, then in each system OD600 is adjusted to about 10 in the exponential phase of standby object.

Then biology was carried out in 100ml shaking flasks within 0,24 and 48 hour period with 200rpm at 37 DEG C to turn Change.Each prepared product is containing 5.0g/l L- homoserine and 12g/l KH₂PO₄[the 200g/l KH of 600 μ l₂PO₄Stoste] with And it is carried out in the 10ml culture medium As of the sulphur source (i.e. NaMC or DMDS) (referring to embodiment 1) of the amount as provided in table 5.Control It (is free of NaMC without any sulphur source and is free of DMDS).

Table 5：Various concentration and the NaMC of reaction time section, the bioconversion results contrast for compareing (no sulphur source) and DMDS

Sequence

Sequence table

<110>Yingchuang Degussa Co., Ltd

<120> WHCB

<130> .

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1137

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> CDS

<222> (1)..(1137)

<223> metX

<400> 1

atg ccc acc ctc gcg cct tca ggt caa ctt gaa atc caa gcg atc ggt 48

Met Pro Thr Leu Ala Pro Ser Gly Gln Leu Glu Ile Gln Ala Ile Gly

1 5 10 15

gat gtc tcc acc gaa gcc gga gca atc att aca aac gct gaa atc gcc 96

Asp Val Ser Thr Glu Ala Gly Ala Ile Ile Thr Asn Ala Glu Ile Ala

20 25 30

tat cac cgc tgg ggt gaa tac cgc gta gat aaa gaa gga cgc agc aat 144

Tyr His Arg Trp Gly Glu Tyr Arg Val Asp Lys Glu Gly Arg Ser Asn

35 40 45

gtc gtt ctc atc gaa cac gcc ctc act gga gat tcc aac gca gcc gat 192

Val Val Leu Ile Glu His Ala Leu Thr Gly Asp Ser Asn Ala Ala Asp

50 55 60

tgg tgg gct gac ttg ctc ggt ccc ggc aaa gcc atc aac act gat att 240

Trp Trp Ala Asp Leu Leu Gly Pro Gly Lys Ala Ile Asn Thr Asp Ile

65 70 75 80

tac tgc gtg atc tgt acc aac gtc atc ggt ggt tgc aac ggt tcc acc 288

Tyr Cys Val Ile Cys Thr Asn Val Ile Gly Gly Cys Asn Gly Ser Thr

85 90 95

gga cct ggc tcc atg cat cca gat gga aat ttc tgg ggt aat cgc ttc 336

Gly Pro Gly Ser Met His Pro Asp Gly Asn Phe Trp Gly Asn Arg Phe

100 105 110

ccc gcc acg tcc att cgt gat cag gta aac gcc gaa aaa caa ttc ctc 384

Pro Ala Thr Ser Ile Arg Asp Gln Val Asn Ala Glu Lys Gln Phe Leu

115 120 125

gac gca ctc ggc atc acc acg gtc gcc gca gta ctt ggt ggt tcc atg 432

Asp Ala Leu Gly Ile Thr Thr Val Ala Ala Val Leu Gly Gly Ser Met

130 135 140

ggt ggt gcc cgc acc cta gag tgg gcc gca atg tac cca gaa act gtt 480

Gly Gly Ala Arg Thr Leu Glu Trp Ala Ala Met Tyr Pro Glu Thr Val

145 150 155 160

ggc gca gct gct gtt ctt gca gtt tct gca cgc gcc agc gcc tgg caa 528

Gly Ala Ala Ala Val Leu Ala Val Ser Ala Arg Ala Ser Ala Trp Gln

165 170 175

atc ggc att caa tcc gcc caa att aag gcg att gaa aac gac cac cac 576

Ile Gly Ile Gln Ser Ala Gln Ile Lys Ala Ile Glu Asn Asp His His

180 185 190

tgg cac gaa ggc aac tac tac gaa tcc ggc tgc aac cca gcc acc gga 624

Trp His Glu Gly Asn Tyr Tyr Glu Ser Gly Cys Asn Pro Ala Thr Gly

195 200 205

ctc ggc gcc gcc cga cgc atc gcc cac ctc acc tac cgt ggc gaa cta 672

Leu Gly Ala Ala Arg Arg Ile Ala His Leu Thr Tyr Arg Gly Glu Leu

210 215 220

gaa atc gac gaa cgc ttc ggc acc aaa gcc caa aag aac gaa aac cca 720

Glu Ile Asp Glu Arg Phe Gly Thr Lys Ala Gln Lys Asn Glu Asn Pro

225 230 235 240

ctc ggt ccc tac cgc aag ccc gac cag cgc ttc gcc gtg gaa tcc tac 768

Leu Gly Pro Tyr Arg Lys Pro Asp Gln Arg Phe Ala Val Glu Ser Tyr

245 250 255

ttg gac tac caa gca gac aag cta gta cag cgt ttc gac gcc ggc tcc 816

Leu Asp Tyr Gln Ala Asp Lys Leu Val Gln Arg Phe Asp Ala Gly Ser

260 265 270

tac gtc ttg ctc acc gac gcc ctc aac cgc cac gac att ggt cgc gac 864

Tyr Val Leu Leu Thr Asp Ala Leu Asn Arg His Asp Ile Gly Arg Asp

275 280 285

cgc gga ggc ctc aac aag gca ctc gaa tcc atc aaa gtt cca gtc ctt 912

Arg Gly Gly Leu Asn Lys Ala Leu Glu Ser Ile Lys Val Pro Val Leu

290 295 300

gtc gca ggc gta gat acc gat att ttg tac ccc tac cac cag caa gaa 960

Val Ala Gly Val Asp Thr Asp Ile Leu Tyr Pro Tyr His Gln Gln Glu

305 310 315 320

cac ctc tcc aga aac ctg gga aat cta ctg gca atg gca aaa atc gta 1008

His Leu Ser Arg Asn Leu Gly Asn Leu Leu Ala Met Ala Lys Ile Val

325 330 335

tcc cct gtc ggc cac gat gct ttc ctc acc gaa agc cgc caa atg gat 1056

Ser Pro Val Gly His Asp Ala Phe Leu Thr Glu Ser Arg Gln Met Asp

340 345 350

cgc atc gtg agg aac ttc ttc agc ctc atc tcc cca gac gaa gac aac 1104

Arg Ile Val Arg Asn Phe Phe Ser Leu Ile Ser Pro Asp Glu Asp Asn

355 360 365

cct tcg acc tac atc gag ttc tac atc taa tag 1137

Pro Ser Thr Tyr Ile Glu Phe Tyr Ile

370 375

<210> 2

<211> 377

<212> PRT

<213> Corynebacterium glutamicum

<400> 2

Met Pro Thr Leu Ala Pro Ser Gly Gln Leu Glu Ile Gln Ala Ile Gly

1 5 10 15

Asp Val Ser Thr Glu Ala Gly Ala Ile Ile Thr Asn Ala Glu Ile Ala

20 25 30

Tyr His Arg Trp Gly Glu Tyr Arg Val Asp Lys Glu Gly Arg Ser Asn

35 40 45

Val Val Leu Ile Glu His Ala Leu Thr Gly Asp Ser Asn Ala Ala Asp

50 55 60

Trp Trp Ala Asp Leu Leu Gly Pro Gly Lys Ala Ile Asn Thr Asp Ile

65 70 75 80

Tyr Cys Val Ile Cys Thr Asn Val Ile Gly Gly Cys Asn Gly Ser Thr

85 90 95

Gly Pro Gly Ser Met His Pro Asp Gly Asn Phe Trp Gly Asn Arg Phe

100 105 110

Pro Ala Thr Ser Ile Arg Asp Gln Val Asn Ala Glu Lys Gln Phe Leu

115 120 125

Asp Ala Leu Gly Ile Thr Thr Val Ala Ala Val Leu Gly Gly Ser Met

130 135 140

Gly Gly Ala Arg Thr Leu Glu Trp Ala Ala Met Tyr Pro Glu Thr Val

145 150 155 160

Gly Ala Ala Ala Val Leu Ala Val Ser Ala Arg Ala Ser Ala Trp Gln

165 170 175

Ile Gly Ile Gln Ser Ala Gln Ile Lys Ala Ile Glu Asn Asp His His

180 185 190

Trp His Glu Gly Asn Tyr Tyr Glu Ser Gly Cys Asn Pro Ala Thr Gly

195 200 205

Leu Gly Ala Ala Arg Arg Ile Ala His Leu Thr Tyr Arg Gly Glu Leu

210 215 220

Glu Ile Asp Glu Arg Phe Gly Thr Lys Ala Gln Lys Asn Glu Asn Pro

225 230 235 240

Leu Gly Pro Tyr Arg Lys Pro Asp Gln Arg Phe Ala Val Glu Ser Tyr

245 250 255

Leu Asp Tyr Gln Ala Asp Lys Leu Val Gln Arg Phe Asp Ala Gly Ser

260 265 270

Tyr Val Leu Leu Thr Asp Ala Leu Asn Arg His Asp Ile Gly Arg Asp

275 280 285

Arg Gly Gly Leu Asn Lys Ala Leu Glu Ser Ile Lys Val Pro Val Leu

290 295 300

Val Ala Gly Val Asp Thr Asp Ile Leu Tyr Pro Tyr His Gln Gln Glu

305 310 315 320

His Leu Ser Arg Asn Leu Gly Asn Leu Leu Ala Met Ala Lys Ile Val

325 330 335

Ser Pro Val Gly His Asp Ala Phe Leu Thr Glu Ser Arg Gln Met Asp

340 345 350

Arg Ile Val Arg Asn Phe Phe Ser Leu Ile Ser Pro Asp Glu Asp Asn

355 360 365

Pro Ser Thr Tyr Ile Glu Phe Tyr Ile

370 375

<210> 3

<211> 1314

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> CDS

<222> (1)..(1314)

<223> metY

<400> 3

atg cca aag tac gac aat tcc aat gct gac cag tgg ggc ttt gaa acc 48

Met Pro Lys Tyr Asp Asn Ser Asn Ala Asp Gln Trp Gly Phe Glu Thr

1 5 10 15

cgc tcc att cac gca ggc cag tca gta gac gca cag acc agc gca cga 96

Arg Ser Ile His Ala Gly Gln Ser Val Asp Ala Gln Thr Ser Ala Arg

20 25 30

aac ctt ccg atc tac caa tcc acc gct ttc gtg ttc gac tcc gct gag 144

Asn Leu Pro Ile Tyr Gln Ser Thr Ala Phe Val Phe Asp Ser Ala Glu

35 40 45

cac gcc aag cag cgt ttc gca ctt gag gat cta ggc cct gtt tac tcc 192

His Ala Lys Gln Arg Phe Ala Leu Glu Asp Leu Gly Pro Val Tyr Ser

50 55 60

cgc ctc acc aac cca acc gtt gag gct ttg gaa aac cgc atc gct tcc 240

Arg Leu Thr Asn Pro Thr Val Glu Ala Leu Glu Asn Arg Ile Ala Ser

65 70 75 80

ctc gaa ggt ggc gtc cac gct gta gcg ttc tcc tcc gga cag gcc gca 288

Leu Glu Gly Gly Val His Ala Val Ala Phe Ser Ser Gly Gln Ala Ala

85 90 95

acc acc aac gcc att ttg aac ctg gca gga gcg ggc gac cac atc gtc 336

Thr Thr Asn Ala Ile Leu Asn Leu Ala Gly Ala Gly Asp His Ile Val

100 105 110

acc tcc cca cgc ctc tac ggt ggc acc gag act cta ttc ctt atc act 384

Thr Ser Pro Arg Leu Tyr Gly Gly Thr Glu Thr Leu Phe Leu Ile Thr

115 120 125

ctt aac cgc ctg ggt atc gat gtt tcc ttc gtg gaa aac ccc gac gac 432

Leu Asn Arg Leu Gly Ile Asp Val Ser Phe Val Glu Asn Pro Asp Asp

130 135 140

cct gag tcc tgg cag gca gcc gtt cag cca aac acc aaa gca ttc ttc 480

Pro Glu Ser Trp Gln Ala Ala Val Gln Pro Asn Thr Lys Ala Phe Phe

145 150 155 160

ggc gag act ttc gcc aac cca cag gca gac gtc ctg gat att cct gcg 528

Gly Glu Thr Phe Ala Asn Pro Gln Ala Asp Val Leu Asp Ile Pro Ala

165 170 175

gtg gct gaa gtt gcg cac cgc aac agc gtt cca ctg atc atc gac aac 576

Val Ala Glu Val Ala His Arg Asn Ser Val Pro Leu Ile Ile Asp Asn

180 185 190

acc atc gct acc gca gcg ctc gtg cgc ccg ctc gag ctc ggc gca gac 624

Thr Ile Ala Thr Ala Ala Leu Val Arg Pro Leu Glu Leu Gly Ala Asp

195 200 205

gtt gtc gtc gct tcc ctc acc aag ttc tac acc ggc aac ggc tcc gga 672

Val Val Val Ala Ser Leu Thr Lys Phe Tyr Thr Gly Asn Gly Ser Gly

210 215 220

ctg ggc ggc gtg ctt atc gac ggc gga aag ttc gat tgg act gtc gaa 720

Leu Gly Gly Val Leu Ile Asp Gly Gly Lys Phe Asp Trp Thr Val Glu

225 230 235 240

aag gat gga aag cca gta ttc ccc tac ttc gtc act cca gat gct gct 768

Lys Asp Gly Lys Pro Val Phe Pro Tyr Phe Val Thr Pro Asp Ala Ala

245 250 255

tac cac gga ttg aag tac gca gac ctt ggt gca cca gcc ttc ggc ctc 816

Tyr His Gly Leu Lys Tyr Ala Asp Leu Gly Ala Pro Ala Phe Gly Leu

260 265 270

aag gtt cgc gtt ggc ctt cta cgc gac acc ggc tcc acc ctc tcc gca 864

Lys Val Arg Val Gly Leu Leu Arg Asp Thr Gly Ser Thr Leu Ser Ala

275 280 285

ttc aac gca tgg gct gca gtc cag ggc atc gac acc ctt tcc ctg cgc 912

Phe Asn Ala Trp Ala Ala Val Gln Gly Ile Asp Thr Leu Ser Leu Arg

290 295 300

ctg gag cgc cac aac gaa aac gcc atc aag gtt gca gaa ttc ctc aac 960

Leu Glu Arg His Asn Glu Asn Ala Ile Lys Val Ala Glu Phe Leu Asn

305 310 315 320

aac cac gag aag gtg gaa aag gtt aac ttc gca ggc ctg aag gat tcc 1008

Asn His Glu Lys Val Glu Lys Val Asn Phe Ala Gly Leu Lys Asp Ser

325 330 335

cct tgg tac gca acc aag gaa aag ctt ggc ctg aag tac acc ggc tcc 1056

Pro Trp Tyr Ala Thr Lys Glu Lys Leu Gly Leu Lys Tyr Thr Gly Ser

340 345 350

gtt ctc acc ttc gag atc aag ggc ggc aag gat gag gct tgg gca ttt 1104

Val Leu Thr Phe Glu Ile Lys Gly Gly Lys Asp Glu Ala Trp Ala Phe

355 360 365

atc gac gcc ctg aag cta cac tcc aac ctt gca aac atc ggc gat gtt 1152

Ile Asp Ala Leu Lys Leu His Ser Asn Leu Ala Asn Ile Gly Asp Val

370 375 380

cgc tcc ctc gtt gtt cac cca gca acc acc acc cat tca cag tcc gac 1200

Arg Ser Leu Val Val His Pro Ala Thr Thr Thr His Ser Gln Ser Asp

385 390 395 400

gaa gct ggc ctg gca cgc gcg ggc gtt acc cag tcc acc gtc cgc ctg 1248

Glu Ala Gly Leu Ala Arg Ala Gly Val Thr Gln Ser Thr Val Arg Leu

405 410 415

tcc gtt ggc atc gag acc att gat gat atc atc gct gac ctc gaa ggc 1296

Ser Val Gly Ile Glu Thr Ile Asp Asp Ile Ile Ala Asp Leu Glu Gly

420 425 430

ggc ttt gct gca atc tag 1314

Gly Phe Ala Ala Ile

435

<210> 4

<211> 437

<212> PRT

<213> Corynebacterium glutamicum

<400> 4

Met Pro Lys Tyr Asp Asn Ser Asn Ala Asp Gln Trp Gly Phe Glu Thr

1 5 10 15

Arg Ser Ile His Ala Gly Gln Ser Val Asp Ala Gln Thr Ser Ala Arg

20 25 30

Asn Leu Pro Ile Tyr Gln Ser Thr Ala Phe Val Phe Asp Ser Ala Glu

35 40 45

His Ala Lys Gln Arg Phe Ala Leu Glu Asp Leu Gly Pro Val Tyr Ser

50 55 60

Arg Leu Thr Asn Pro Thr Val Glu Ala Leu Glu Asn Arg Ile Ala Ser

65 70 75 80

Leu Glu Gly Gly Val His Ala Val Ala Phe Ser Ser Gly Gln Ala Ala

85 90 95

Thr Thr Asn Ala Ile Leu Asn Leu Ala Gly Ala Gly Asp His Ile Val

100 105 110

Thr Ser Pro Arg Leu Tyr Gly Gly Thr Glu Thr Leu Phe Leu Ile Thr

115 120 125

Leu Asn Arg Leu Gly Ile Asp Val Ser Phe Val Glu Asn Pro Asp Asp

130 135 140

Pro Glu Ser Trp Gln Ala Ala Val Gln Pro Asn Thr Lys Ala Phe Phe

145 150 155 160

Gly Glu Thr Phe Ala Asn Pro Gln Ala Asp Val Leu Asp Ile Pro Ala

165 170 175

Val Ala Glu Val Ala His Arg Asn Ser Val Pro Leu Ile Ile Asp Asn

180 185 190

Thr Ile Ala Thr Ala Ala Leu Val Arg Pro Leu Glu Leu Gly Ala Asp

195 200 205

Val Val Val Ala Ser Leu Thr Lys Phe Tyr Thr Gly Asn Gly Ser Gly

210 215 220

Leu Gly Gly Val Leu Ile Asp Gly Gly Lys Phe Asp Trp Thr Val Glu

225 230 235 240

Lys Asp Gly Lys Pro Val Phe Pro Tyr Phe Val Thr Pro Asp Ala Ala

245 250 255

Tyr His Gly Leu Lys Tyr Ala Asp Leu Gly Ala Pro Ala Phe Gly Leu

260 265 270

Lys Val Arg Val Gly Leu Leu Arg Asp Thr Gly Ser Thr Leu Ser Ala

275 280 285

Phe Asn Ala Trp Ala Ala Val Gln Gly Ile Asp Thr Leu Ser Leu Arg

290 295 300

Leu Glu Arg His Asn Glu Asn Ala Ile Lys Val Ala Glu Phe Leu Asn

305 310 315 320

Asn His Glu Lys Val Glu Lys Val Asn Phe Ala Gly Leu Lys Asp Ser

325 330 335

Pro Trp Tyr Ala Thr Lys Glu Lys Leu Gly Leu Lys Tyr Thr Gly Ser

340 345 350

Val Leu Thr Phe Glu Ile Lys Gly Gly Lys Asp Glu Ala Trp Ala Phe

355 360 365

Ile Asp Ala Leu Lys Leu His Ser Asn Leu Ala Asn Ile Gly Asp Val

370 375 380

Arg Ser Leu Val Val His Pro Ala Thr Thr Thr His Ser Gln Ser Asp

385 390 395 400

Glu Ala Gly Leu Ala Arg Ala Gly Val Thr Gln Ser Thr Val Arg Leu

405 410 415

Ser Val Gly Ile Glu Thr Ile Asp Asp Ile Ile Ala Asp Leu Glu Gly

420 425 430

Gly Phe Ala Ala Ile

435

<210> 5

<211> 41

<212> DNA

<213> Hybrid promoter

<220>

<221> promoter

<222> (1)..(41)

<223> PtacI

<400> 5

gagctgttga caattaatca tcggctcgta taatgtgtgg a 41

<210> 6

<211> 3255

<212> DNA

<213> synthesized sequence

<400> 6

cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 60

tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 120

accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 180

gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 240

tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcggca cacagcccag 300

cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 360

ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attatggagc tgttgacaat 420

taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 480

agaattcaaa ggaggacaac catgcccacc ctcgcgcctt caggtcaact tgaaatccaa 540

gcgatcggtg atgtctccac cgaagccgga gcaatcatta caaacgctga aatcgcctat 600

caccgctggg gtgaataccg cgtagataaa gaaggacgca gcaatgtcgt tctcatcgaa 660

cacgccctca ctggagattc caacgcagcc gattggtggg ctgacttgct cggtcccggc 720

aaagccatca acactgatat ttactgcgtg atctgtacca acgtcatcgg tggttgcaac 780

ggttccaccg gacctggctc catgcatcca gatggaaatt tctggggtaa tcgcttcccc 840

gccacgtcca ttcgtgatca ggtaaacgcc gaaaaacaat tcctcgacgc actcggcatc 900

accacggtcg ccgcagtact tggtggttcc atgggtggtg cccgcaccct agagtgggcc 960

gcaatgtacc cagaaactgt tggcgcagct gctgttcttg cagtttctgc acgcgccagc 1020

gcctggcaaa tcggcattca atccgcccaa attaaggcga ttgaaaacga ccaccactgg 1080

cacgaaggca actactacga atccggctgc aacccagcca ccggactcgg cgccgcccga 1140

cgcatcgccc acctcaccta ccgtggcgaa ctagaaatcg acgaacgctt cggcaccaaa 1200

gcccaaaaga acgaaaaccc actcggtccc taccgcaagc ccgaccagcg cttcgccgtg 1260

gaatcctact tggactacca agcagacaag ctagtacagc gtttcgacgc cggctcctac 1320

gtcttgctca ccgacgccct caaccgccac gacattggtc gcgaccgcgg aggcctcaac 1380

aaggcactcg aatccatcaa agttccagtc cttgtcgcag gcgtagatac cgatattttg 1440

tacccctacc accagcaaga acacctctcc agaaacctgg gaaatctact ggcaatggca 1500

aaaatcgtat cccctgtcgg ccacgatgct ttcctcaccg aaagccgcca aatggatcgc 1560

atcgtgagga acttcttcag cctcatctcc ccagacgaag acaacccttc gacctacatc 1620

gagttctaca tctaatagac gcgtgagctg ttgacaatta atcatcggct cgtataatgt 1680

gtggaattgt gagcggataa caatttcacg cgtttaatta acacgagtac tggaaaacta 1740

aatgccaaag tacgacaatt ccaatgctga ccagtggggc tttgaaaccc gctccattca 1800

cgcaggccag tcagtagacg cacagaccag cgcacgaaac cttccgatct accaatccac 1860

cgctttcgtg ttcgactccg ctgagcacgc caagcagcgt ttcgcacttg aggatctagg 1920

ccctgtttac tcccgcctca ccaacccaac cgttgaggct ttggaaaacc gcatcgcttc 1980

cctcgaaggt ggcgtccacg ctgtagcgtt ctcctccgga caggccgcaa ccaccaacgc 2040

cattttgaac ctggcaggag cgggcgacca catcgtcacc tccccacgcc tctacggtgg 2100

caccgagact ctattcctta tcactcttaa ccgcctgggt atcgatgttt ccttcgtgga 2160

aaaccccgac gaccctgagt cctggcaggc agccgttcag ccaaacacca aagcattctt 2220

cggcgagact ttcgccaacc cacaggcaga cgtcctggat attcctgcgg tggctgaagt 2280

tgcgcaccgc aacagcgttc cactgatcat cgacaacacc atcgctaccg cagcgctcgt 2340

gcgcccgctc gagctcggcg cagacgttgt cgtcgcttcc ctcaccaagt tctacaccgg 2400

caacggctcc ggactgggcg gcgtgcttat cgacggcgga aagttcgatt ggactgtcga 2460

aaaggatgga aagccagtat tcccctactt cgtcactcca gatgctgctt accacggatt 2520

gaagtacgca gaccttggtg caccagcctt cggcctcaag gttcgcgttg gccttctacg 2580

cgacaccggc tccaccctct ccgcattcaa cgcatgggct gcagtccagg gcatcgacac 2640

cctttccctg cgcctggagc gccacaacga aaacgccatc aaggttgcag aattcctcaa 2700

caaccacgag aaggtggaaa aggttaactt cgcaggcctg aaggattccc cttggtacgc 2760

aaccaaggaa aagcttggcc tgaagtacac cggctccgtt ctcaccttcg agatcaaggg 2820

cggcaaggat gaggcttggg catttatcga cgccctgaag ctacactcca accttgcaaa 2880

catcggcgat gttcgctccc tcgttgttca cccagcaacc accacccatt cacagtccga 2940

cgaagctggc ctggcacgcg cgggcgttac ccagtccacc gtccgcctgt ccgttggcat 3000

cgagaccatt gatgatatca tcgctgacct cgaaggcggc tttgctgcaa tctagggccg 3060

gccgtttaaa ccctgcaggt ccgggacctg caggcatgca agcttggcac tggccgtcgt 3120

tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 3180

tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3240

gttgcgcagc ctgaa 3255

201500228 A 2

Claims

1. the method for producing l-methionine, wherein culture has L- Kosé in the culture medium comprising L- homoserine and sulphur source Propylhomoserin O- acetyltransferase activities and the active microorganism of O- acetyl-L- sulfhydrylases, the sulphur source are selected from first The salt and dimethyl disulphide of mercaptan, methyl mercaptan, thus accumulate l-methionine in the medium.

2. according to the method described in claim 1, the wherein described microorganism is recombination, and L- homoserine O- acetyl shifts Enzymatic activity and O- acetyl-L- sulfhydrylase activity are enhancings.

3. according to the method described in claim 2, the wherein described L- homoserine O- acetyltransferase activities and O- acetyl-L- are high Serine sulfhydrylase activity is enhanced by increased gene expression, wherein the gene code has L- homoserine The protein or coding of O- acetyltransferase activities have the active protein of O- acetyl-L- sulfhydrylases.

4. method according to claim 3, wherein the increased gene expression is by increasing the copy number of the gene by reality It is existing, wherein protein and/or coding of the gene code with L- homoserine O- acetyltransferase activities are with O- acetyl- The active protein of L- sulfhydrylases.

5. method according to claim 3 or 4, wherein the increased gene expression is by starting the gene with strong Sub-functionality connect and realizes, wherein the gene code have L- homoserine O- acetyltransferase activities protein with/ Or coding has the active protein of O- acetyl-L- sulfhydrylases.

6. the wherein described microorganism is selected from enterobacteriaceae and bar bacterium the method according to any one of claims 1 to 5, Section.

7. method according to any one of claim 1 to 6, wherein the L- homoserine O- acetyltransferase activities are MetX enzymes from Corynebacterium glutamicum (Corynebacterium glutamicum).

8. method according to any one of claim 1 to 7, wherein O- acetyl-L- sulfhydrylases are active It is derived from the MetY enzymes of Corynebacterium glutamicum (Corynebacterium glutamicum).